The invention is generally in the field of infection, inflammation and allergy. More specifically, the present invention concerns three novel DNA molecules encoding three polypeptides, which may be useful in controlling and modulating activation and differentiation of lymphoid cells. The present invention also concerns expression vectors comprising the genes, host cells comprising the expression vectors, proteins produced by the genes, methods for producing the proteins, and methods of using the genes and proteins.
Natural killer (NK) cells are lymphocytes that participate in innate immune response against certain bacteria, parasites, and viruses. (Lanier, L. L., (1998) Annu. Rev. Immunol. 16:359-393). NK cells express a lectin-like receptor superfamily of type II transmembrane proteins (amino terminus intracellular). Their extracellular domains have structural features of C-type lectins. (Ryan, J. C., et al., (1997) Immunol. Rev. 155:79-89). The superfamily consists of several families including Ly-49 (in mice and rats), NKR-P1 (in mice, rats, and humans), NKG2 (in humans and rats), and CD94 (in humans). These proteins are encoded by a single genetic region called the NK gene complex (NKC) which are located on human chromosome 12, mouse chromosome 6 and rat chromosome 4. Different receptors, even within the same family, have been shown to activate or to inhibit NK cell functions. (Vely, F., et al., (1997) J. Immunol. 159:2075-2077). In many cases, the different activities mediated by individual receptors have been linked to the different structures of these receptors in their cytoplasmic domain and in their transmembrane domain.
For example, the murine Ly-49D and Ly-49H, and the human NKG2C, which possess a positively charged residue (arginine orlysine) within their transmembrane domain, have been shown to activate NK cells by associating with DAP12 membrane adapter protein. (Smith, K. M., et al., (1998) J. Immunol. 161:7-10; Lanier, L. L., et al., (1998) Immunity 8:693-701). The DAP12 contains a negatively charged residue (aspartic acid) in its transmembrane region and an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. Upon cross-linking of CD94/NKG2C, tyrosine residues in ITAM of DAP12 become phosphorylated and recruit tyrosine kinases, such as ZAP-70 or Syk.
On the other hand, the murine Ly-49A that lacks charged residues in its transmembrane region and contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain has been demonstrated to inhibit NK cytotoxicity. (Nakamura, M. C., et al., (1997) J. Exp. Med. 185:673-684). The inhibitory activity is mediated by cytoplasmic tyrosine phosphatase, SHP-1, which is recruited by the ITIM domain of Ly-49A. The tyrosine phosphatase SHP-1 can dephosphorylate the adjacent adapter proteins and kinases, resulting in the termination of activation signals.
Genes located in NKC also encode other C-type lectins such as CD69 and the recently identified receptor AICL. (Lopez-Cabrera, M., et al., (1993) J. Exp. Med. 178:537-547; Hamann, J., et al., (1997) Immunogenetics 45:295-300). Unlike the restricted expression of other NK cell receptors, both CD69 and AICL are widely expressed on hematopoietic cells including lymphocytes, monocytes and granulcytes. They are not expressed on resting, but are rapidly induced upon activation. CD69 is known as the earliest activation marker of lymphocytes. Anti-CD69 mAb can induce activation and cytokine production of T, B and NK cells, though CD69 lacks a charged residue in its intracellular domain. (Testi, R., et al., (1994) Immunol Today. 15:479-483).
Because of the diverse biological activities of members of the lectin-like receptor superfamily and their close relationship to immune cell functions, those skilled in the art are interested in identifying novel members of this family. The identification and study of novel genes and proteins may lead to a better understanding of the mechanisms underlying immune cell functions, and will permit those skilled in the art to regulate or control immune reactions or diseases.
The present invention includes a novel xe2x80x9cCLAXxe2x80x9d protein (C-type Lectin, Activation eXpressed) shown in SEQ ID NO:2 (FIG. 2A) and a nucleic acid sequence (SEQ ID NO:1) encoding said CLAX protein. Additionally encompassed within the invention are nucleic acid sequences encoding homologues to said CLAX protein (FIGS. 2B, 2C and 2D). The homologues are referred to herein as clone 7B (nucleic acid sequence shown in SEQ ID NO:3; amino acid sequence shown in SEQ ID NO:4); clone 2I (nucleic acid sequence shown in SEQ ID NO:5; amino acid sequence shown in SEQ ID NO:6); and clone 4A (nucleic acid sequence shown in SEQ ID NO:7; amino acid sequence shown in SEQ ID NO:8). The nucleotide sequences of the isolated cDNA""s are disclosed herein along with the deduced amino acid sequences. The cDNA genes of the above clones have been deposited on Jan. 19, 1999 with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 and given the Accession Numbers ATCC 203599 (HuCLAX-7B) (clone 7B); ATCC 203601 (HuCLAX-2I) (clone 2I); and ATCC 203600 (HuCLAX-4A) (clone 4A).
The present inventors sequenced the clones encoding the novel CLAX protein homologues and determined the primary sequences of the deduced proteins. The nucleic acid and amino acid sequences of the novel CLAX protein disclosed herein were determined from the sequenced clones. The novel CLAX protein exhibits sequence identity to the known sequence of human CD69.
The CLAX protein of the present invention can be produced by: (1) inserting the cDNA of a disclosed CLAX into an appropriate expression vector; (2) transfecting the expression vector into an appropriate transfection host(s); (3) growing the transfected host(s) in appropriate culture media; and (4) purifying the protein from the culture media.
The present invention therefore provides a purified and isolated nucleic acid molecule, preferably a DNA molecule, having a sequence which codes for CLAX protein, or an oligonucleotide fragment of the nucleic acid molecule which is unique to a CLAX protein of the present invention. In a preferred embodiment of the invention, the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:1 (FIG. 2A). In another preferred embodiment, the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:3 (FIG. 2B). In still another preferred embodiment the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:5 (FIG. 2C). In still another preferred embodiment of the present invention the purified and isolated nucleic acid molecule has the nucleotide sequence as shown in SEQ ID NO:7 (FIG. 2D).
The invention also contemplates a double stranded nucleic acid molecule comprising a nucleic acid molecule of the invention or an oligonucleotide fragment thereof hydrogen bonded to a complementary nucleotide base sequence.
The terms xe2x80x9cisolated and purified nucleic acidxe2x80x9d and xe2x80x9csubstantially pure nucleic acidxe2x80x9d, e.g., substantially pure DNA, refer to a nucleic acid molecule which is one or both of the following: (1) not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally occurring genome of the organism from which the nucleic acid is derived; or (2) which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure or isolated and purified DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional CLAX sequence.
The present invention provides in one embodiment: (a) an isolated and purified nucleic acid molecule comprising a sequence encoding all or a portion of a protein having the amino acid sequence as shown in SEQ ID NO:2; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which exhibit at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions. In a particular embodiment, the nucleic acid sequence comprises (a) the sequence as shown in SEQ ID NO:1, (b) a nucleic acid sequence complementary to SEQ ID NO:1, and (c) sequences having at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a) or (b).
The degree of homology (percent identity) between a native and a mutant sequence may be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose. One suitable program is the GAP computer program described by Devereux et al., (1984) Nucl. Acids Res. 12:387. The GAP program utilizes the alignment method of Needleman and Wunsch (1970) J. Mol. Biol. 48:433, as revised by Smith and Waterman (1981) Adv. Appl. Math. 2:482. Briefly, the GAP program defines percent identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences.
As used herein the term xe2x80x9cstringent conditionsxe2x80x9d encompasses conditions known in the art under which a nucleotide sequence will hybridize to an isolated and purified nucleic acid molecule comprising a sequence encoding a protein having the amino acid sequence as shown herein, or to (b) a nucleic acid sequence complementary to (a). In a preferred embodiment, stringent conditions comprise overnight incubation at 42xc2x0 C. in a solution comprising: 50% formamide, 5xc3x97SSPE (750 mM NaCl, 50 mM NaH2PO4 and 5 mM EDTA), 5xc3x97Denhardt""s solution, 0.1% SDS and 100 xcexcg/ml denatured, sheared salmon sperm DNA. One skilled in the art may vary conditions appropriately. Screening polynucleotides under stringent conditions may be carried out according to the method described in Nature, 313:402-404 (1985). Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present invention may be, for example, allelic variants of the disclosed DNA sequences, or may be derived from other sources. General techniques of nucleic acid hybridization are disclosed by Sambrook et al., xe2x80x9cMolecular Cloning: A Laboratory Manualxe2x80x9d, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1984); and by Haymes et al., xe2x80x9cNucleic Acid Hybridization: A Practical Approachxe2x80x9d, IRL Press, Washington, D.C. (1985), which references are incorporated herein by reference.
The present invention provides in another embodiment: (a) an isolated and purified nucleic acid molecule comprising a sequence encoding all or a portion of a protein having the amino acid sequence as shown in SEQ ID NO:4 (clone 7B; FIG. 2B); (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.
The present invention provides in another embodiment: (a) an isolated and purified nucleic acid molecule comprising a sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:6 (clone 2I; FIG. 2C); (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.
The present invention provides in another embodiment: (a) an isolated and purified nucleic acid molecule comprising a sequence encoding all or a portion of a protein having the amino acid sequence as shown in SEQ ID NO:8 (clone 4A; FIG. 2D); (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.
The present invention also provides: (a) a purified and isolated nucleic acid molecule comprising a sequence as shown in SEQ ID NO:1 (FIG. 2A); (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.
The present invention further provides: (a) a purified and isolated nucleic acid molecule comprising a sequence as shown in SEQ ID NO:3 (clone 7B; FIG. 2B); (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.
The present invention further provides: (a) a purified and isolated nucleic acid molecule comprising a sequence as shown in SEQ ID NO:5 (clone 2I; FIG. 2C); (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.
The present invention further provides: (a) a purified and isolated nucleic acid molecule comprising a sequence as shown in SEQ ID NO:7 (clone 4A; FIG. 2D); (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.
The present invention additionally covers nucleic acid and amino acid molecules of the present invention having one or more structural mutations including replacement, deletion or insertion mutations. For example, a signal peptide may be deleted, or conservative amino acid substitutions may be made to generate a protein that is still biologically competent or active.
The invention further contemplates a recombinant molecule comprising a nucleic acid molecule of the present invention or an oligonucleotide fragment thereof and an expression control sequence operatively linked to the nucleic acid molecule or oligonucleotide fragment. A transformant host cell including a recombinant molecule of the invention is also provided.
In another aspect, the invention features a cell or purified preparation of cells which include a novel gene encoding a CLAX protein of the present invention, or which otherwise misexpresses a gene encoding a CLAX protein of the present invention. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a CLAX transgene, e.g., a heterologous form of a CLAX gene, e.g., a gene derived from humans (in the case of a non-human cell). The CLAX transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpresses an endogenous CLAX gene, e.g., a gene that increases expression of an endogenous CLAX gene, or a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or misexpressed CLAX alleles for use in drug screening.
Still further, the invention provides plasmids that comprise the nucleic acid molecules of the invention.
The present invention also includes a novel CLAX of the present invention, or an active part thereof. A biologically competent or active form of the protein or part thereof is also referred to herein as an xe2x80x9cactive CLAX or part thereofxe2x80x9d.
The invention further contemplates antibodies having specificity against an epitope of the CLAX protein of the present invention, or part of the protein. These antibodies may be polyclonal or monoclonal. The antibodies may be labeled with a detectable substance and they may be used, for example, to detect the novel CLAX of the invention in tissue and cells. Additionally, the antibodies of the present.invention, or portions thereof, may be used to make targeted antibodies that destroy CLAX expressing cells (e.g., antibody-toxin fusion proteins, or radiolabelled antibodies).
The invention also permits the construction of nucleotide probes that encode part or all of the novel CLAX protein of the invention or a part of the protein. Thus, the invention also relates to a probe comprising a nucleotide sequence coding for a protein, which displays the properties of the novel CLAX of the invention or a peptide unique to the protein. The probe may be labeled, for example, with a detectable (e.g., radioactive) substance and it may be used to select from a mixture of nucleotide sequences a nucleotide sequence coding for a protein-which displays the properties of the novel CLAX protein of the invention.
The present invention also provides a transgenic non-human animal (e.g., a rodent, e.g., a mouse or a rat, a rabbit or a pig) or embryo all of whose germ cells and somatic cells contain a recombinant molecule of the invention, preferably a recombinant molecule comprising a nucleic acid molecule of the present invention encoding a CLAX of the invention or part thereof. The recombinant molecule may comprise a nucleic acid sequence encoding the CLAX of the present invention with a structural mutation, or may comprise a nucleic acid sequence encoding the CLAX protein of the invention or part thereof and one or more regulatory elements which differ from the regulatory elements that drive expression of the native protein. In another preferred embodiment, the animal has a CLAX gene that is misexpressed (e.g., over-expressed) or not expressed (e.g., a knockout). Such transgenic animals can serve as models for studying disorders that are related to mutated or misexpressed CLAX of the present invention.
The invention still further provides a method for identifying a substance which is capable of binding to and/or modulating the novel CLAX of the present invention, said method comprising reacting the novel CLAX of the invention or part of the protein under conditions which permit the formation of a complex between the substance and the novel CLAX protein or part of the protein, and assaying for substance-CLAX complexes, for free substance, for non-complexed CLAX, or for activation of the CLAX.
An embodiment of the invention provides a method for identifying ligands which are capable of binding to the novel CLAX protein of the invention, isoforms thereof, or part of the protein, said method comprising reacting the novel CLAX protein of the invention, isoforms thereof, or part of the protein, with at least one ligand which potentially is capable of binding to the protein, isoform, or part of the protein, under conditions which permit the formation of ligand-receptor protein complexes, and assaying for ligand-receptor protein complexes, for free ligand, for non-complexed CLAX protein, or for activation of the CLAX protein. In a preferred embodiment of the method, ligands are identified which are capable of binding to and activating or inactivating the novel CLAX protein of the invention, isoforms thereof, or part(s) of the protein.
The invention also relates to a method for assaying a medium for the presence of an agonist or antagonist of the interaction of the novel CLAX protein and a substance which is capable of binding the CLAX, said method comprising providing a known concentration of a CLAX protein, reacting the CLAX with a substance which is capable of binding to the CLAX and a suspected agonist or antagonist under conditions which permit the formation of substance-CLAX complexes, and assaying for substance-CLAX complexes, for free substance, for non-complexed CLAX, or for activation of the CLAX protein.
The invention further provides a method for identifying a substance which is capable of binding to an activated CLAX protein of the present invention or an isoform or a part of the protein, said method comprising reacting an activated CLAX of the present invention, or an isoform or part of the protein, with at least one substance which potentially can bind with the CLAX, isoform or part of the protein, under conditions which permit the formation of substance-activated CLAX complexes, and assaying for substance-CLAX complexes, for free substance, or for non-complexed CLAX. The method may be used to identify intracellular ligands containing proteins that bind to an activated CLAX protein of the present invention or parts thereof, or intracellular ligands that may be affected in other ways by the activated CLAX of the invention.
Also included within the scope of the present invention is a composition which includes a CLAX of the present invention, a fragment thereof (or a nucleic acid encoding said CLAX or fragment thereof) and, optionally, one or more additional components, e.g., a carrier, diluent or solvent. The additional component can be one that renders the composition useful for in vitro, in vivo, pharmaceutical or veterinary use.
In another aspect, the present invention relates to a method of treating a mammal, e.g., a human, at risk for a disorder, e.g., a disorder characterized by aberrant or unwanted level or biological activity of the CLAX of the present invention, or characterized by an aberrant or unwanted level of a ligand that specifically binds to a CLAX of the present invention. For example, the CLAX of the present invention may be useful to leach out or block a ligand which is found to bind to the CLAX of the present invention. Encompassed within the scope of the invention is a soluble form of the CLAX protein of the present invention, e.g., a fragment of the receptor, that may be used to inhibit activation of the receptor by binding to the ligand a polypeptide of the present invention and preventing the ligand from interacting with membrane bound CLAX.
Also within the scope of the present invention are fusion proteins comprising all or a portion of the CLAX of the present invention. Preferably, the fusion protein comprises all or a portion of the extracellular region of the CLAX of the present invention as shown in SEQ ID NO:2. All or a portion of the extracellular portion of the CLAX of the present invention may be attached to another molecule or polypeptide, e.g., a hinge and/or constant region of an immunoglobulin (xe2x80x9cIgxe2x80x9d) protein. Also included within the present invention are soluble fusion proteins comprising all or a portion of CLAX, and additionally comprising an extracellular domain of another receptor molecule (e.g., an extracellular domain of murine CD8 at the N-terminal side and an extracellular domain of CLAX at the C-terminal side). Examples of soluble fusion proteins are given in FIG. 4.
The primary object of the present invention is the identification of a new human CLAX, as identified by its sequence disclosed herein. Additional objects of the invention are the methods of using the cDNA, the CLAX protein, a monoclonal antibody specific for the novel CLAX, fusion proteins comprising a portion of the CLAX protein of the present invention, and a ligand for the novel CLAX as described above.